Advertisement

Protoplasma

, Volume 253, Issue 2, pp 611–623 | Cite as

The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility

  • Micah Rapp
  • Jessica Schein
  • Kevin A. Hunt
  • Vamsi Nalam
  • George S. Mourad
  • Neil P. SchultesEmail author
Original Article

Abstract

The solute specificity profiles (transport and binding) for the nucleobase cation symporter 1 (NCS1) proteins, from the closely related C4 grasses Zea mays and Setaria viridis, differ from that of Arabidopsis thaliana and Chlamydomonas reinhardtii NCS1. Solute specificity profiles for NCS1 from Z. mays (ZmNCS1) and S. viridis (SvNCS1) were determined through heterologous complementation studies in NCS1-deficient Saccharomyces cerevisiae strains. The four Viridiplantae NCS1 proteins transport the purines adenine and guanine, but unlike the dicot and algal NCS1, grass NCS1 proteins fail to transport the pyrimidine uracil. Despite the high level of amino acid sequence similarity, ZmNCS1 and SvNCS1 display distinct solute transport and recognition profiles. SvNCS1 transports adenine, guanine, hypoxanthine, cytosine, and allantoin and competitively binds xanthine and uric acid. ZmNCS1 transports adenine, guanine, and cytosine and competitively binds, 5-fluorocytosine, hypoxanthine, xanthine, and uric acid. The differences in grass NCS1 profiles are due to a limited number of amino acid alterations. These amino acid residues do not correspond to amino acids essential for overall solute and cation binding or solute transport, as previously identified in bacterial and fungal NCS1, but rather may represent residues involved in subtle solute discrimination. The data presented here reveal that within Viridiplantae, NCS1 proteins transport a broad range of nucleobase compounds and that the solute specificity profile varies with species.

Keywords

Setaria viridis Zea mays Purine Pyrimidine Nucleobase cation symporter 1 

Notes

Acknowledgments

We thank Regan Huntley and Carol Clark at The Connecticut Agricultural Experiment Station for expert technical assistance. We also thank the Biology Department at the University of Saint Francis, Fort Wayne, Indiana, for use of their confocal microscope. This work was funded by research funds from IPFW to G.S.M. and Hatch Fund CONH00253 to N.P.S.

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

709_2015_838_MOESM1_ESM.docx (59 kb)
ESM 1 Alignment of NCS1 proteins from Viridiplantae by ClustalW (Thompson et al. 1994). AcNCS1 Aquilegia coerulea Aquca_009_01069.1; AlNCS1, Arabidopsis lyrata XP_002873103; AtNCS1, Arabidopsis thaliana NP_568122.2; BdNCS1B, Brachypodium distachyon Bradi3g14360.1; BdNCS1A, Brachypodium distachyon Bradi3g51350.1; CarNCS1, Capsella rubella Carubv10003714m; CcNCS1, Citrus clementina Ciclev10011424m; CisNCS1, Citrus sinensis orange1.1g009276m; CrNCS1, Chlamydomonas reinhardtii XP_001694932.1; CsNCS1, Cucumis sativus Cucsa.378830.1; FvNCS1, Fragaria vesca mrna00956.1-v1.0-hybrid; GmNCS1A, Glycine max Glyma13g21131.1; GmNCS1B, Glycine max Glyma10g07230.1i; GrNCS1A, Gossypium raimondii Gorai.006G160100.1; GrNCS1B, Gossypium raimondii Gorai.006G160000.1; HvNCS1, Hordeum vulgare BAJ85216.1; LuNCS1, Linum usitatissimum LuS10014397; MdNCS1, Malus domestica MDP0000253445; MeNCS1, Manihot esculenta cassava4.1_007082m; MgNCS1, Mimulus guttatus mgv1a003893m; MpCCMP1545NCS1, Micromonas pusilla CCMP15451594 MicpuC2.gw1.11.67.1; MpRCC299NCS1, Micromonas pusilla RCC299 55664 EuGene.0200010041; MtNCS1A, Medicago truncatula Medtr7g102810.1; MtNCS1B, Medicago truncatula Medtr1g062130.1; NsNCS1, Nicotiana sylvestris (Schultes & Mourad pers. Comm); ObNCS1, Oryza brachyantha XP_006647657.1; OlNCS1, Ostreococcus lucimarinus 49824|estExt_Genewise_ext.C_Chr_60346; OsNCS1, Oryza sativa LOC_Os02g44680.1; OtNCS1, Ostreococcus tauri Ostta_3|19171|estExt_fgenesh1_pg.C_chrom_06.10330; PgNCS1, Picea glauca gb|BT114389.1; PpNCS1A, Physcomitrella patens Pp1s34_250V6.1 (Phypa_120934); PpNCS1B, Physcomitrella patens Pp1S171_59V6.1 (Phypa_191990); PtNCS1, Populus trichocarpa Potri.006G119500.1; PvNCS1, Phaseolus vulgaris Phvul.007G226300.1; PvNCS1A, Panicum virgatum Pavirv00007850m; PvNCS1B, Panicum virgatum Pavirv00030059m; RcNCS1, Ricinus communis 30078.m002226; SbNCS1, Sorghum bicolor Sb04g032390.1; SiNCS1B, Setaria italica Si016858m; SiNCS1A, Setaria italica Si019168m; SmNCS1, Selaginella moellendorffii gene 93539; StNCS1, Solanum tuberosum XP_006354397; SvNCS1, Setaria viridis AHC53692.1; TcNCS1, Theobroma cacao Thecc1EG022354t1; ThNCS1, Thellungiella halophila Thhalv10015725m; VcNCS1, Volvox carteri Vocar20015267m; VvNCS1, Vitus vinifera GSVIVT01033705001; ZmNCS1, Zea mays GRMZM2G362848_T01. Transmembrane domains for ZmNCS1 are labeled as given in Figure 1 legend. Underlined text in italics represents the predicted chloroplast transit sequence and cleavage site given by ChloroP (Emanuelsson et al. 1999) for ZmNCS1 and SvNCS1. Text in red (with * below) denotes amino acid identity among NCS1 presented, while text in blue (with . below) or green (with : below) denotes strong and weak amino acid similarities, respectively, among NCS1 proteins presented as given by ClustalW. Bold black italic text highlighted with gray background in ZmNCS1 identifies amino acid sequence present in proteomic analysis of maize chloroplast membranes (Friso et al. 2010). The twenty-nine amino acid differences between SvNCS1 and ZmNCS1, past the predicted chloroplast cleavage site of SvNCS1, are as identified in Figure 1 legend. Bold black numbers identify amino acids that differ between SvNCS1 and ZmNCS1 and are not conserved among other NCS1. Bold white numbers with black background identify amino acid differences between SvNCS1 and ZmNCS1 but are predominantly conserved among all other NCS1 presented. Text in the AtNCS1 sequence with aqua background identifies amino acid sites mutated by site-directed mutagenesis (Witz et al. 2014) (DOCX 59 kb)
709_2015_838_Fig9_ESM.gif (46 kb)
ESM 2

Time course for uptake of radiolabeled nucleobase of SvNCS1 and ZmNCS1 in S. cerevisiae. Uptake of 1.0 μM [8-3H]-hypoxanthine in S. cerevisiae strains containing SvNCS1 (pCC207) over time (a), of 0.5 μM [2,8-3H]-adenine in S. cerevisiae strain (fcy2∆) containing SvNCS1 (pCC207) (b) or ZmNCS1 (pNS487) (c) over time (GIF 46 kb)

709_2015_838_MOESM2_ESM.tif (100 kb)
High resolution image (TIFF 100 kb)

References

  1. Argyrou E, Sophianopoulou V, Schultes N, Diallinas G (2001) Functional characterization of a maize purine transporter by expression in Aspergillus nidulans. Plant Cell 13:953–964CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ashihara H, Sano H, Crozier A (2008) Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. Phytochemistry 69:841–856CrossRefPubMedGoogle Scholar
  3. Bennetzen JL, Schmutz J, Wang H, Percifield R, Hawkins J, Pontaroli AC, Estep M, Feng L, Vaughn JN, Grimwood J, Jenkins J, Barry K, Lindquist E, Hellsten U, Deshpande S, Wang X, Wu X, Mitros T, Triplett J, Yang X, Ye C-Y, Mauro-Herrera M, Wang L, Li P, Manoj Sharma M, Sharma R, Ronald PC, Panaud O, Kellogg EA, Brutnell TP, Doust AN, Tuskan GA, Rokhsar D, Devos KM (2012) Reference genome sequence of the model plant Setaria. Nat Biotechnol 30:555–561CrossRefPubMedGoogle Scholar
  4. Bréthes D, Chirio MC, Napias C, Chevallier MR, Lavie JL, Chevallier J (1992) In vivo and in vitro studies of the purine cytosine permease of Saccharomyces cerevisiae: functional analysis of a mutant with an altered apparent transport constant of uptake. Eur J Biochem 204:699–704CrossRefPubMedGoogle Scholar
  5. Bürkle L, Cedzich A, Dopke C, Stransky H, Okumoto S, Gillissen B, Kuhn C, Frommer WB (2003) Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis. Plant J 34:13–26CrossRefPubMedGoogle Scholar
  6. Cedzich A, Stransky H, Schulz B, Frommer WB (2008) Characterization of cytokinin and adenine transport in Arabidopsis cell cultures. Plant Physiol 148:1857–1867CrossRefPubMedPubMedCentralGoogle Scholar
  7. Christianson TW, Sikorsk RS, Dant M, Shero JH, Hieter P (1992) Multifunctional yeast high copy-number shuttle vectors. Gene 110:119–122CrossRefPubMedGoogle Scholar
  8. Collier R, Tegeder M (2012) Soybean ureide transporters play a critical role in nodule development, function and nitrogen export. Plant J 72:355–367CrossRefPubMedGoogle Scholar
  9. De Koning H, Diallinas G (2000) Nucleobase transporters. Mol Membr Biol 17:75–94CrossRefPubMedGoogle Scholar
  10. Desimone M, Catoni E, Ludewig U, Hilpert M, Schneider A, Kunze R, Tegeder M, Frommer WB, Schumacher K (2002) A novel superfamily of transporters for allantoin and other oxo-derivatives of nitrogen heterocyclic compounds in Arabidopsis. Plant Cell 14:847–856CrossRefPubMedPubMedCentralGoogle Scholar
  11. Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984CrossRefPubMedPubMedCentralGoogle Scholar
  12. Ferreira T, Bréthes D, Pinson B, Napias C, Chevallier J (1997) Functional analysis of mutated purine-cytosine permease from Saccharomyces cerevisiae. J Biol Chem 272:9697–9702CrossRefPubMedGoogle Scholar
  13. Ferreira T, Napias C, Chevallier J, Bréthes D (1999a) Evidence for a dynamic role for proline376 in the purine-cytosine permease of Saccharomyces cerevisiae. Eur J Biochem 263:57–64CrossRefPubMedGoogle Scholar
  14. Ferreira T, Chevallier J, Paumard P, Napias C, Bréthes D (1999b) Screening of an intragenic second-site suppressor of purine-cytosine permease from Saccharomyces cerevisiae. Eur J Biochem 260:22–30CrossRefPubMedGoogle Scholar
  15. Frébort I, Kowalska M, Hluska T, Frébortova J, Galuszka P (2011) Evolution of cytokinin biosynthesis and degradation. J Exp Bot 62:2431–2452CrossRefPubMedGoogle Scholar
  16. Friso G, Majeran W, Huang M, Sun Q, van Wijk KJ (2010) Reconstruction of metabolic pathways, protein expression, and homeostasis machineries across maize bundle sheath and mesophyll chloroplasts: large-scale quantitative proteomics using the first maize genome assembly1. Plant Physiol 152:1219–1250CrossRefPubMedPubMedCentralGoogle Scholar
  17. Gietz DR, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96CrossRefPubMedGoogle Scholar
  18. Gillissen B, Bürkle L, André B, Kühn C, Rentsch K, Brandl B, Frommer WB (2000) A new family of high-affinity transporters for adenine, cytosine, and purine derivatives in Arabidopsis. Plant Cell 12:291–300CrossRefPubMedPubMedCentralGoogle Scholar
  19. Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten U, Putnam N, Daniel S, Rokhsar DS (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 40(D1):D1178–D1186CrossRefPubMedPubMedCentralGoogle Scholar
  20. Gournas C, Papageorgiou I, Diallinas G (2008) The nucleobase-ascorbate transporter (NAT) family: genomics, evolution, structure-function relationships and physiological role. Mol Biosyst 4:404–414CrossRefPubMedGoogle Scholar
  21. Hamari Z, Amillis S, Drevet C, Apostolaki A, Vágvölgyi C, Diallinas G, Scazzocchio C (2009) Convergent evolution and orphan genes in the Fur4p-like family and characterization of a general nucleoside transporter in Aspergillus nidulans. Mol Microbiol 73:43–57CrossRefPubMedGoogle Scholar
  22. Jelesko JG (2012) An expanding role for purine uptake permease-like transporters in plant secondary metabolism. Front Plant Sci 3:1–5CrossRefGoogle Scholar
  23. Jeschke G (2013) A comparative study of structures and structural transitions of secondary transporters with the LeuT fold. Eur Biophys J 42:181–197CrossRefPubMedPubMedCentralGoogle Scholar
  24. Jund R, Chevallier MR, Lacroute F (1988) Primary structure of the uracil transport protein of Saccharomyces cerevisiae. Eur J Biochem 171:417–424CrossRefPubMedGoogle Scholar
  25. Kafer C, Zhou L, Santoso D, Guirgis A, Weers B, Park S, Thornburg R (2004) Regulation of pyrimidine metabolism in plants. Front Biosci 9:1611–1625CrossRefPubMedGoogle Scholar
  26. Kombrick E, Beevers H (1983) Transport of purine and pyrimidine bases and nucleosides from endosperm to cotyledons in germinating castor bean seedlings. Plant Physiol 73:370–376CrossRefGoogle Scholar
  27. Krogh A, Larsson B, von Heijne G, Sonnhammer ELL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580CrossRefPubMedGoogle Scholar
  28. Krypotou E, Kosti V, Amillis S, Myrianthopoulos V, Mikros E, Diallinas G (2012) Modeling, substrate docking and mutational analysis identify residues essential for the function and specificity of a eukaryotic purine-cytosine NCS1 transporter. J Biol Chem 287:36792–36803CrossRefPubMedPubMedCentralGoogle Scholar
  29. Ma P, Baldwin JM, Baldwin SA, Henderson PJF (2013) Membrane transport proteins: the nucleobase cation symporter 1 family. In: Roberts GCK (ed) Encyclopedia of biophysics. Springer, European Biophysical Societies’ Association, pp 1485–1489CrossRefGoogle Scholar
  30. Mansfield TA, Schultes NP, Mourad GS (2009) AtAzg1 and AtAzg2 comprise a novel family of purine transporters in Arabidopsis. Fed Euro Biochem Soc Lett 583:481–486CrossRefGoogle Scholar
  31. Maurino VG, Grube E, Zielinski J, Schild A, Fischer K, Flügge U-I (2006) Identification and expression analysis of twelve members of the nucleobase–ascorbate transporter (NAT) gene family in Arabidopsis thaliana. Plant Cell Physiol 47:1381–1393CrossRefPubMedGoogle Scholar
  32. Moffatt BA, Ashihara H (2002) Purine and pyrimidine nucleotide synthesis and metabolism In: Somerville CR, Meyerowitz EM (eds) The Arabidopsis book. American Society of Plant Biologists, pp 1-20. doi/ 10.1199/tab.0018
  33. Mourad GS, Tippmann-Crosby J, Hunt KA, Gicheru Y, Bade K, Mansfield TA, Schultes NP (2012) Genetic and molecular characterization reveals a unique nucleobase cation symporter 1 in Arabidopsis. Fed Euro Biochem Soc Lett 586:1370–1378CrossRefGoogle Scholar
  34. Nelson BK, Cai X, Nebenführ A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136CrossRefPubMedGoogle Scholar
  35. Niopek-Witz S, Deppe J, Lemieux MJ, Möhlmann T (2014) Biochemical characterization and structure-function relationship of two plant NCS2 proteins, the nucleobase transporters NAT3 and NAT12 from Arabidopsis thaliana. Biochim Biophys Acta 12:3025–3035CrossRefGoogle Scholar
  36. Pantazopoulou A, Diallinas G (2007) Fungal nucleobase transporters. FESM Microbiol Rev 31:657–675CrossRefGoogle Scholar
  37. Ross CW (1991) Biosynthesis of nucleotides, In: Stumpf PK, Conn EE (eds) The biochemistry of plants. Academic Press, pp 169–205.Google Scholar
  38. Schein J, Hunt KA, Minton J, Schultes NP, Mourad GS (2013) The nucleobase cation symporter 1 from Chlamydomonas reinhardtii and the evolutionary distant Arabidopsis thaliana share function and establish a plant-specific solute transport profile. Plant Physiol Biochem 70:52–60CrossRefPubMedGoogle Scholar
  39. Schmidt A, Su Y_H, Kunze R, Warner S, Hewitt M, Slocum RD, Ludewig U, Frommer WB, Desimone M (2004) UPS1 and UPS2 from Arabidopsis mediate high affinity transport of uracil and 5-fluorouracil. J Biol Chem 279:44817–44824CrossRefPubMedGoogle Scholar
  40. Schmidt A, Baumann N, Schwarzkopf A, Frommer WB, Desimone M (2006) Comparative studies on ureide permeases in Arabidopsis thaliana and analysis of two alternative splice variants of AtUPS5. Planta 224:1329–1340CrossRefPubMedGoogle Scholar
  41. Schultes NP, Brutnell TP, Allen A, Dellaporta SL, Nelson T, Chen J (1996) Leaf permease1 gene of maize is required for chloroplast development. Plant Cell 8:463–475CrossRefPubMedPubMedCentralGoogle Scholar
  42. Serrano R, Villalba J-M (1995) Expression and localization of plant membrane proteins in Saccharomyces. Methods Cell Biol 50:481–496CrossRefPubMedGoogle Scholar
  43. Shi Y (2013) Common folds and transport mechanisms of secondary active transporters. Annu Rev Biophys 42:51–72CrossRefPubMedGoogle Scholar
  44. Shimamura T, Weyand S, Beckstein O, Rutherford NG, Hadden JM, Sharples D, Sansom MSP, Iwata S, Henderson PJF, Cameron AD (2010) Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1. Science 328:470–473CrossRefPubMedPubMedCentralGoogle Scholar
  45. Soderlund C, Descour A, Kudrna D, Bomhoff M, Boyd L, Currie J, Angelova A, Collura K, Wissotski M, Ashley E, Morrow D, Fernandes J, Walbot V, Yu Y (2009) Sequencing, mapping, and analysis of 27,455 maize full-length cDNAs. PLoS Genet 5:e1000740CrossRefPubMedPubMedCentralGoogle Scholar
  46. Stasolla C, Katahira R, Thorpe TA, Ashihara H (2003) Purine and pyrimidine nucleotide metabolism in higher plants. J Plant Physiol 160:1271–1295CrossRefPubMedGoogle Scholar
  47. Stolz J, Vielreicher M (2003) Tpn1p, the plasma membrane vitamin B6 transporter of Saccharomyces cerevisiae. J Biol Chem 278:18990–18996CrossRefPubMedGoogle Scholar
  48. Szydlowski N, Bürkle L, Pourcel L, Moulin M, Stolz J, Fitzpatrick TB (2013) Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis. Plant J 75:40–52CrossRefPubMedGoogle Scholar
  49. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefPubMedPubMedCentralGoogle Scholar
  50. Wagner R, de Montigny J, de Wergifosse P, Souciet JL, Potier S (1998) The ORF YBL042 of Saccharomyces cerevisiae encodes a uridine permease. FESM Microbiol Lett 159:69–75CrossRefGoogle Scholar
  51. Werner AK, Witte C-P (2011) The biochemistry of nitrogen mobilization: purine ring catabolism. Trends Plant Sci 16:381–387CrossRefPubMedGoogle Scholar
  52. Weyand S, Shimamura T, Yajima S, Suzuki S, Mirza O, Krusong K, Carpenter EP, Rutherford NG, Hadden JM, O’Reilly J, Ma P, Saidijam M, Patching SG, Hope RJ, Norbertczak HT, Roach PCJ, Iwata S, Henderson PJF, Cameron AD (2008) Structure and molecular mechanism of a nucleobase–cation–symport-1 family transporter. Science 322:709–713CrossRefPubMedPubMedCentralGoogle Scholar
  53. Weyand S, Shimamura T, Beckstein O, Sansom MSP, Iwata S, Henderson PJF, Cameron AD (2011) The alternating access mechanism of transport as observed in the sodium-hydantoin transporter Mhp1. J Synchrotron Radiat 18:20–23CrossRefPubMedPubMedCentralGoogle Scholar
  54. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, Bakkoury ME, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Davis RW (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906CrossRefPubMedGoogle Scholar
  55. Witz S, Jung B, Fürst S, Möhlmann T (2012) De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis. Plant Cell 24:1549–1559CrossRefPubMedPubMedCentralGoogle Scholar
  56. Witz S, Panwar P, Schober M, Deppe J, Pasha FA, Lemieux MJ, Möhlmann T (2014) Structure-function relationship of a plant NCS1 member—homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis. PLoS 9(3):e91343CrossRefGoogle Scholar
  57. Yan N (2013) Structural investigations of the proton-coupled secondary transporters. Curr Opin Struct Biol 23:483–491CrossRefPubMedGoogle Scholar
  58. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572CrossRefPubMedGoogle Scholar
  59. Zrenner R, Stitt M, Sonnewald U, Boldt R (2006) Pyrimidine and purine biosynthesis and degradation in plants. Annu Rev Plant Biol 57:805CrossRefPubMedGoogle Scholar
  60. Zrenner R, Riegler H, Marquard CR, Lange PR, Geserick C, Bartosz CE, Chen CT, Solcum RD (2009) A functional analysis of the pyrimidine catabolic pathway in Arabidopsis. New Phytol 183:117–132CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • Micah Rapp
    • 1
  • Jessica Schein
    • 1
  • Kevin A. Hunt
    • 1
  • Vamsi Nalam
    • 1
  • George S. Mourad
    • 1
  • Neil P. Schultes
    • 2
    Email author
  1. 1.Department of BiologyIndiana University-Purdue University Fort WayneFort WayneUSA
  2. 2.Department of Plant Pathology and EcologyThe Connecticut Agricultural Experiment StationNew HavenUSA

Personalised recommendations